XIX International Bile Acid Meeting
Bile
Portal blood
NTCP
BA
OATP
OA
OCT
OC
MDR1 MDR2/3
CFTR
PS?
Cl-
FIC1
PL
H2O
Cl-
OA
AQP
BSEP
ABCG5/8
Hepatocyte
MRPs
AE
HCO3-
Bile
ASBT
flow
PC
BA
Ch
Cl-
MRP2
BA
Cholangiocyte
Hepatocyte
Canaliculus
ABCG5/8
BA
PC
MDR3
Cholesterol
Other
OA
BSEP
MRP2
Micelle
BSEP
No BA
No PC
MDR3
MRP2
NTCP
BA
OATP
OA
OCT
OC
MDR1 MDR2/3
CFTR
PS?
Cl-
FIC1
PL
H2O
Cl-
OA
AQP
BSEP
ABCG5/8
Hepatocyte
MRPs
AE
HCO3-
Bile
ASBT
flow
PC
BA
Ch
Cl-
MRP2
BA
Cholangiocyte
uptake
BA
SHP
BA
FXR
RXRα
BA Synthesis
Basolateral
efflux
BSEP staining
Normal liver
BSEP staining
BSEP
deficiency
Malignancy in BSEP deficiency
‹ With
hepatocyte phenotype
– 13 HCC
– Related to cell turnover?
– Related to DNA damage?
– But not related to background AFP
‹ With
cholangiocyte phenotype
– 2 cholangiocarcinomaa
– Poor bile flow?
– Regeneration?
BSEP diseases
‹ ABCB11
mutated in the form of PFIC known
as BSEP deficiency
‹ Mutations
also identified in a collection of
BRIC and ICP patients
Potential disease mechanisms
‹ Missense
mutations can cause:
- abnormal mRNA processing or stability
BRCA1, NFB1
- improper protein processing and trafficking
CFTR, MRP2, α1AT def
- defects in protein function
CFTR, MRP2
mRNA processing or stability
‹ Native
1
α-globin
exons and
introns
in vitro minigene system:
2
3 -1
transfection into
cells
+1 3
fibronectin
NBD exon and
intron
spliced mRNA
product
Pagani F et al. J Biol Chem
2000;275:21041-7
‹ Investigative
in vitro minigene
system:
1
2
3 -1
transfection into
cells
exon and
introns
+1 3
of interest
spliced mRNA
product containing
exon of interest
‹ RT-PCR
analysis of in vitro minigene system:
738 bp
550 bp
238 bp
no
product
exon spliced into
minigene
differentially spliced
exon
exon ‘skipped’ from native
minigene
unstable minigene
Strategy for ABCB11
‹-
wild type ABCB11 exons and 500 bp of intron
‹ 62
missense mutations
‹ 14
SNPs
- minigenes into HepG2 cells and RT-PCR and
sequencing
Protein results
‹
11 Mutations associated with immature processing
‹
16 Mutations associated with no protein
Protein results
‹
6 Mutations with probable functional defects:
1 SNP has higher level of protein than wild type
‹
3 SNPs have same protein levels as wild type
Summary
‹
Bile acid transport is essential!
‹
Failure of bile acid transport leads to severe liver disease
and malignancy
‹
62 missense mutations and SNPs in human ABCB11 have
been classified
‹
This may allow the development of mutation-specific
therapies
King’s College London
Jane Byrne
Sandra Strautnieks
Alex Knisely
The body of man has in itself
blood, phlegm, yellow bile, and
black bile………. ….pain is felt
when one of these elements is
in defect or excess……...